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Atomic Details of the Interactions of Glycosaminoglycans with Amyloid-# Fibrils Katie L. Stewart, Eleri Hughes, Edwin Alexander Yates, Teng-Yi Huang, Marcelo A. Lima, Timothy R. Rudd, Marco Guerrini, Shang-Cheng Hung, Sheena E Radford, and David A. Middleton J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 9, 2016
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Journal of the American Chemical Society
Atomic Details of the Interactions of Glycosaminoglycans with Amyloid-β Fibrils Katie L. Stewart,† Eleri Hughes,‡ Edwin A. Yates,§ Teng-Yi Huang,◊ Marcelo A. Lima,Ⱶ Timothy R. Rudd,# Marco Guerrini,∆ Shang-Cheng Hung,§* Sheena E. Radford,†* and David A. Middleton‡* †
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, University of Leeds, Leeds
LS2 9JT, United Kingdom ‡
Department of Chemistry, University of Lancaster, Lancaster LA1 4YB, United Kingdom
§
Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom
◊ Ⱶ
Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei, Taiwan
Department of Biochemistry, Federal University of Sao Paulo (UNIFESP), Rua Três de Maio, São Paulo, 40440-020 Brazil
#
National Institute of Biological Standards and Control (NIBSC), South Mimms, Potters Bar, Hertfordshire EN6 3QC, Unit-
ed Kingdom ∆
Ronzoni Institute for Chemical and Biochemical Research, Via G. Colombo 81, Milano 20133 Italy
Supporting Information Placeholder ABSTRACT: The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-β (Aβ) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aβ self-assembly and can impede fibril clearance and therefore may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aβ fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aβ β fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly 13C-labeled octasaccharide heparin analog enabled magic-angle spinning solidstate NMR (SSNMR) of the GAG bound to 3Q fibrils, and measurements of dynamics reveal a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular 13C-15N dipolar dephasing is consistent with close (< 5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aβ40 fibrils and a major non-protein component of AD plaques, and reveal that GAGamyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture.
Alzheimer’s disease (AD) is characterized pathologically by the accumulation of insoluble plaques within the extracellular spaces of brain tissue. The main protein constituents of AD plaques are the 40- and 42-residue amyloid-β peptides, Aβ40 and Aβ42, derived from the amyloid precursor protein. The relationship between Aβ self-assembly and disease has not been elucidated, but the development of drugs that lower the propensity of Aβ peptides
to self-assemble or reduce the aggregate concentration is a therapeutic goal.1 Aβ amyloid plaques in the brain are highly heterogeneous, comprising fibrous proteins of different structural organisations2, metal ions, nucleic acids and glycosaminoglycans (GAGs),3 the linear sulfated polysaccharide components of proteoglycans. GAGs accelerate Aβ polymerization4 and increase fibril resistance to proteolytic degradation.5 The recent clinical failure of drugs that inhibit Aβ aggregation in vitro6 may reflect, in part, the role of molecules such as GAGs in the development and stabilization of amyloid in the brain. Work focusing on GAGs has revealed a complicated picture of interactions with protein networks, with low binding specificity and high redundancy among heterogeneous saccharide units7. We previously detected a GAG binding site at the surface of fibrillar MAβ40 (an Aβ40 homologue with an N-terminal methionine) from heparin-induced peptide chemical shift perturbations measured using cross-polarization magic-angle spinning (CPMAS) solid-state NMR (SSNMR).8 In this and previous studies, heparin was employed as a proxy for the highly sulfated domain of heparan sulfate (HS) commonly associated with amyloid plaques and sharing the same disaccharide units as heparin.9. Aβ40 fibrils are polymorphic and fibril strains having approximate 2-fold (2A) or 3-fold (3Q) cross-sectional symmetry can be selected by seeding and morphologically confirmed by SSNMR.8,10 Previously, we developed an assay to quantify heparin binding which involves sedimentation of bound heparin with fibrils, and quantifies unbound heparin by addition of heparinase enzyme, creating a spectroscopically active product.8 We found that heparin binds with surprisingly higher affinity to MAβ40 3Q fibrils than to MAβ40 2A fibrils, and the SSNMR data suggests that heparin recognizes the junctions of the triangular crosssection that is unique in the structures of 3Q fibrils (Figure S1).8
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Figure 1. GAG binding to amyloid fibrils. (A) Binding affinities of fibrils to low molecular weight heparin (LMWH, dp16). Curves were obtained by non-linear least-squares fitting of a Hill function. (B) Apparent dissociation constants Kd for MAβ40 3Q fibrils binding heparin fragments of different degrees of polymerization (dp4-dp18). (C) TEM image of 3Q fibrils co-sedimented with [U13 C]OHA. (D) Chemical structure of [U-13C]OHA.
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Figure 2. 13C CP-MAS SSNMR spectra of the [U-15N] 3Q fibril[U-13C]OHA complex. (A) Experimental spectrum (black) at 4°C, with assignments, and a simulated spectrum from the solution chemical shifts (red). (B) 2D symmetrized 13C-13C spectrum recorded with a DARR mixing time of 20 ms (black) and an unsymmetrized spectrum at 50 ms mixing time (red).
Here we show, in addition, that de novo MAβ40 fibrils assembled without seeding, a peptide comprised of Aβ residues 16-22 (KLVFFAE), human amylin (hIAPP) which is 47% similar in sequence to Aβ40, and a uniform morphology of MAβ42 fibrils 11 bind weakly to low-molecular weight heparin (LMWH) (Figure 1A, Figure S2). These binding differences suggest a unique relationship between 3Q fibrils in particular and heparin, but the origin of this specific relationship requires a full molecular-level understanding of the 3Q fibril-heparin interaction. Whether all heparin residues lie in contact with specific amino acids (Figure S1, top), or whether part of heparin lies away from the fibril surface (Figure S1, bottom) is not known. Here, synthesis of a uniformly 13C-labelled octasaccharide heparin analog ([U-13C]OHA) has enabled the detailed analysis of a GAG-amyloid interaction for the first time from the GAG perspective, using crosspolarization magic-angle spinning (CP-MAS) SSNMR to observe the GAG bound to insoluble 3Q MAβ40 fibrils.
tion and the pellet, estimated to contain 600 nmoles MAβ40 and 170 nmoles bound [U-13C]OHA, was analysed by 13C CP-MAS SSNMR, revealing signature peaks (60-102 ppm) from the octasaccharide. Under CP-MAS, signals are detected only from nuclei in environments where molecular dynamics are too slow ( 10 ppm) and ~231 ppm and 181 ppm for the conjugate base.18 Here the peak at 161 ppm may reflect the stabilization of protonated His ring(s) by the octasaccharide CO2- and SO3- groups. A non-selective 15N-observed, 13C-dephased rotational-echo double-resonance (15N{13C}REDOR) experiment (Figure 3E) showed weak dephasing of peptide amide resonances (112-130 ppm) and possibly also of His resonances, although the signal-to-noise is rather poor. Dipolar dephasing of resonances assigned to Arg, Lys and Gly was not observed above the noise. Dephasing was not quantified because of uncertainties arising from there being a 3-fold excess of fibrils over [U-13C]OHA and because of small contributions from 13C at natural abundance. A 13 C-observed frequency-selective rotational-echo doubleresonance (FSR) experiment19 was used to detect specific 15N-13C dipolar interactions between His side-group 15Nδ1/ε2 sites and saccharide C-1 carbons in the 97-100 ppm region. The observed dipolar dephasing (S/S0 = 0.72 ± 0.15), corresponds to a 13C-15N 3
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repeating side groups (~4.7 Å) (Figure 4B). This, and our binding data on other fibril types (Figure 1A) suggest that GAG-3Q interactions are more specific than mere electrostatic patterning. Instead, the structure of the 3Q fibril corners localizes multiple charged and hydrogen bonding residues in a favorable orientation, providing a tight and specific binding site for heparin (Figure S11). The involvement of histidine residues in this interaction suggests that disease states manifesting local pH changes could enhance GAG-fibril interactions, leading to altered rates of disease progression. Fibrils with different binding modes and affinities may rationalize the array of phenotypes that are present in AD and the range of timescales over which the disease progresses.
Prof. A. Wilson for Aβ40 16-22. The UK 850 MHz solid-state NMR Facility was funded by EPSRC, BBSRC, and the University of Warwick, including funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands and the European Regional Development Fund.
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Figure 4. Model of [U-13C]OHA binding to MAβ40 fibrils. (A) Cross-sectional view of the 3Q fibril, from 2lmq.pdb (negative stagger, with 9 flexible N-terminal residues added) viewed down the fibril axis (z) and highlighting key binding residues and OHA (purple spheres) docked in one of the three cleft sites. (B) View of an octasaccharide molecule with repeating H6 and N27 residues. The work presented provides the first glimpse of a GAG chain binding tightly and offers rationalization for the quantifiable and surprisingly specific affinity for a well-defined morphology of Aβ40 fibrils. A three-fold conformation of Aβ has been isolated and purified from human brain tissue, underscoring the relevance of this structural motif in disease2. These results reveal that while GAGs are commonly found associated with amyloid plaques, GAG-fibril interactions show remarkable specificity which may influence the varied properties of amyloid fibrils in disease.
ASSOCIATED CONTENT Supporting Information Additional information as noted in the text and details of all preparative and experimental methods. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Authors Email:
[email protected];
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[email protected];
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT The BBSRC (UK) for grants BB/K01451X/1 and BB/K015958/1. Academia Sinica and Ministry of Science and Technology (MOST 104-0210-01-09-02 & 104-2628-M-001-001). We thank Dr R. Tycko for providing the fibril seeds and Dr. G. Preston and 4
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